Nucleic acids encoding antibody molecules which bind IL-17A and IL-17F

ABSTRACT

The invention relates to antibody molecules having specificity for antigenic determinants of both IL-17A and IL-17F, therapeutic uses of the antibody molecules and methods for producing said antibody molecules.

This application is divisional of U.S. application Ser. No. 12/446,143,filed Jun. 11, 2010, which is a national phase entry of InternationalApplication No. PCT/GB2007/003983, filed Oct. 18, 2007, now U.S. Pat.No. 8,303,953, which claims the benefit of Great Britain Application No.0620729.4, filed Oct. 18, 2006, the disclosures of which areincorporated by reference herein.

The present invention relates to antibody molecules having specificityfor antigenic determinants of both IL-17A and IL-17F. The presentinvention also relates to the therapeutic uses of the antibody moleculesand methods for producing them.

Interleukin 17 (IL-17), also known as CTLA-8 or IL-17A, is apro-inflammatory cytokine which stimulates the secretion of a wide rangeof other cytokines from various non-immune cells. IL-17A is capable ofinducing the secretion of IL-6, IL-8, PGE2, MCP-1 and G-CSF by adherentcells like fibroblasts, keratinocytes, epithelial and endothelial cellsand is also able to induce ICAM-1 surface expression, proliferation of Tcells, and growth and differentiation of CD34+ human progenitors intoneutrophils when cocultured in the presence of irradiated fibroblasts(Fossiez et al., 1998, Int. Rev. Immunol. 16, 541-551). IL-17A ispredominantly produced by activated memory T cells and acts by bindingto a ubiquitously distributed cell surface receptor (IL-17R) (Yao etal., 1997, Cytokine, 9, 794-800). It may also act through binding to acomplex of IL-17RA and IL-17RC (Toy et al., 2006, J. Immunol. 177(11);36-39). IL-17 producing T cells called ‘TH17 cells’ have been implicatedin the pathogenesis of certain cancers (Weaver et al., 2006, Immunity,24, 677-688; Langowski et al., 2006, 442, 461-465; Iwakura and Ishigame,2006, J. Clin. Invest. 116, 5, 1218-1222).

A number of homologues of IL-17 have been identified which have bothsimilar and distinct roles in regulating inflammatory responses. For areview of IL-17 cytokine/receptor families see Dumont, 2003, ExpertOpin. Ther. Patents, 13, 287-303. One such homologue is IL-17F, alsoknown as IL-24 and ML-1, which is around 55% identical to IL-17A and isthought to share the same receptors as IL-17A (Kolls and Linden 2004,Immunity, 21, 467-476; Hymowitz, et al., 2001, EMBO J. 20(19),5332-5341; Kuestner et al., 2007, Journal of Immunology, 179,5462-5473).

Both IL-17A and IL-17F can form both homodimeric and heterodimericproteins, all of which are produced by activated human CD4+ T cells(Wright et al., 2007, J Biol. Chem. 282 (18), 13447-13455).

IL-17 may contribute to a number of diseases mediated by abnormal immuneresponses, such as rheumatoid arthritis and air-way inflammation, aswell as organ transplant rejection and antitumour immunity. Inhibitorsof IL-17 activity are well known in the art for example a murineIL-17R:human Fc fusion protein, a murine soluble IL-17R and ananti-IL-17 monoclonal antibody have been used to demonstrate the role ofIL-17 in various models of rheumatoid arthritis (Lubberts et al., J.Immunol. 2001, 167, 1004-1013; Chabaud et al., Arthritis Res. 2001, 3,168-177). In addition, neutralising polyclonal antibodies have been usedto reduce peritoneal adhesion formation (Chung et al., 2002, J. Exp.Med., 195, 1471-1478). Rat derived anti-human IL-17 antibodies weredescribed in WO04/106377. A humanised anti-IL-17 antibody with anaffinity of around 220 pM was described in WO2006/054059. A monoclonalanti-IL-17 fully human antibody with an affinity of around 188 pM wasdescribed in WO2006/013107. Antibodies which bind IL-17F andIL-17A/IL-17F heterodimers were described in WO2006/088833. Antibodieswhich specifically bind the IL-17A/IL-17F heterodimer were described inWO2005/010044.

IL-17F antagonism has been associated with protection against asthma(Kawaguchi et al., 2006, J. Allergy Clin. Immunol. 117(4); 795-801) andIL-17F is also thought to play a role in arthritis pathology (Lubberts2003, Current Opinion in Investigational Drugs, 4 (5), 572-577).

Accordingly dual antagonists of IL-17A and IL-17F may be more effectivethan a sole antagonist in treating IL-17 mediated diseases. Antibodieswhich bind IL-17A and IL-17F were described in WO2007/106769 published20.9.07.

We have been able to demonstrate that it is possible to isolate anantibody which is capable of binding to both IL-17A and IL-17F and iscapable of neutralising the activity of both isoforms of IL-17. Hencethe present invention provides an anti-IL-17 antibody which is capableof binding to both IL-17A and IL-17F. In particular, the antibody of thepresent invention is capable of specifically binding to both IL-17A andIL-17F i.e. the antibody does not bind to other isoforms of IL-17.Preferably the antibody of the present invention also binds theIL-17A/IL-17F heterodimer. Preferably, the antibody of the presentinvention neutralises the activity of both IL-17A and IL-17F. In oneembodiment the antibody of the present invention also neutralises theactivity of the IL-17A/IL-17F heterodimer. The antibodies of the presentinvention therefore have the advantageous property that they can inhibitthe biological activity of both IL-17A and IL-17F. Accordingly, thepresent invention also provides the use of such antibodies in thetreatment of and/or prophylaxis of a disease mediated by either or bothof IL-17A or IL-17F such as autoimmune or inflammatory disease orcancer.

As used herein, the term ‘neutralising antibody’ describes an antibodythat is capable of neutralising the biological signalling activity ofboth IL-17A and IL17F for example by blocking binding of IL-17A andIL17F to one or more of their receptors and by blocking binding of theIL-17A/IL-17F heterodimer to one or more of its receptors. It will beappreciated that the term ‘neutralising’ as used herein refers to areduction in biological signalling activity which may be partial orcomplete. Further, it will be appreciated that the extent ofneutralisation of IL-17A and IL-17F activity by the antibody may be thesame or different. In one embodiment the extent of neutralisation of theactivity of the IL-17A/IL-17F heterodimer may be the same or differentas the extent of neutralisation of IL-17A or IL-17F activity.

In one embodiment the antibodies of the present invention specificallybind to IL-17A and IL-17F. Specifically binding means that theantibodies have a greater affinity for IL-17A and IL-17F polypeptides(including the IL-17A/IL-17F heterodimer) than for other polypeptides.Preferably the IL-17A and IL-17F polypeptides are human. In oneembodiment the antibody also binds cynomolgus IL-17F.

IL-17A or IL-17F polypeptides or a mixture of the two or cellsexpressing one or both of said polypeptides can be used to produceantibodies which specifically recognise both polypeptides. The IL-17polypeptides (IL-17A and IL-17F) may be ‘mature’ polypeptides orbiologically active fragments or derivatives thereof which preferablyinclude the receptor binding site. Preferably the IL-17 polypeptides arethe mature polypeptides. IL-17 polypeptides may be prepared by processeswell known in the art from genetically engineered host cells comprisingexpression systems or they may be recovered from natural biologicalsources. In the present application, the term “polypeptides” includespeptides, polypeptides and proteins. These are used interchangeablyunless otherwise specified. The IL-17 polypeptide may in some instancesbe part of a larger protein such as a fusion protein for example fusedto an affinity tag. Antibodies generated against these polypeptides maybe obtained, where immunisation of an animal is necessary, byadministering the polypeptides to an animal, preferably a non-humananimal, using well-known and routine protocols, see for example Handbookof Experimental Immunology, D. M. Weir (ed.), Vol 4, BlackwellScientific Publishers, Oxford, England, 1986). Many warm-bloodedanimals, such as rabbits, mice, rats, sheep, cows or pigs may beimmunized. However, mice, rabbits, pigs and rats are generallypreferred.

Antibodies for use in the present invention include whole antibodies andfunctionally active fragments or derivatives thereof and may be, but arenot limited to, monoclonal, multi-valent, multi-specific, humanized orchimeric antibodies, domain antibodies e.g. VH, VL, VHH, single chainantibodies, Fab fragments, Fab′ and F(ab′)₂ fragments andepitope-binding fragments of any of the above. Other antibody fragmentsinclude those described in International patent applicationsWO2005003169, WO2005003170 and WO2005003171. Antibody fragments andmethods of producing them are well known in the art, see for exampleVerma et al., 1998, Journal of Immunological Methods, 216, 165-181;Adair and Lawson, 2005. Therapeutic antibodies. Drug DesignReviews—Online 2(3):209-217.

Antibodies for use in the present invention include immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e. molecules that contain an antigen binding site thatspecifically binds an antigen. The immunoglobulin molecules of theinvention can be of any class (e.g. IgG, IgE, IgM, IgD and IgA) orsubclass of immunoglobulin molecule.

Monoclonal antibodies may be prepared by any method known in the artsuch as the hybridoma technique (Kohler & Milstein, 1975, Nature,256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., 1983, Immunology Today, 4:72) and the EBV-hybridomatechnique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

Antibodies for use in the invention may also be generated using singlelymphocyte antibody methods by cloning and expressing immunoglobulinvariable region cDNAs generated from single lymphocytes selected for theproduction of specific antibodies by for example the methods describedby Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA93(15):7843-78481; WO92/02551; WO2004/051268 and International PatentApplication number WO2004/106377.

Humanized antibodies are antibody molecules from non-human specieshaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework region from a human immunoglobulinmolecule (see, e.g. U.S. Pat. No. 5,585,089; WO91/09967).

Chimeric antibodies are those antibodies encoded by immunoglobulin genesthat have been genetically engineered so that the light and heavy chaingenes are composed of immunoglobulin gene segments belonging todifferent species. These chimeric antibodies are likely to be lessantigenic. Bivalent antibodies may be made by methods known in the art(Milstein et al., 1983, Nature 305:537-539; WO 93/08829, Traunecker etal., 1991, EMBO J. 10:3655-3659). Multi-valent antibodies may comprisemultiple specificities or may be monospecific (see for example WO92/22853 and WO05/113605).

The antibodies for use in the present invention can also be generatedusing various phage display methods known in the art and include thosedisclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50),Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough etal. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 1879-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) and WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.Techniques for the production of single chain antibodies, such as thosedescribed in U.S. Pat. No. 4,946,778 can also be adapted to producesingle chain antibodies which bind to IL-17A and IL-17F. Also,transgenic mice, or other organisms, including other mammals, may beused to express humanized antibodies.

The residues in antibody variable domains are conventionally numberedaccording to a system devised by Kabat et al. This system is set forthin Kabat et al., 1987, in Sequences of Proteins of ImmunologicalInterest, US Department of Health and Human Services, NIH, USA(hereafter “Kabat et al. (supra)”). This numbering system is used in thepresent specification except where otherwise indicated.

The Kabat residue designations do not always correspond directly withthe linear numbering of the amino acid residues. The actual linear aminoacid sequence may contain fewer or additional amino acids than in thestrict Kabat numbering corresponding to a shortening of, or insertioninto, a structural component, whether framework or complementaritydetermining region (CDR), of the basic variable domain structure. Thecorrect Kabat numbering of residues may be determined for a givenantibody by alignment of residues of homology in the sequence of theantibody with a “standard” Kabat numbered sequence.

The CDRs of the heavy chain variable domain are located at residues31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3)according to the Kabat numbering system. However, according to Chothia(Chothia, C. and Lesk, A. M. J. Mol. Biol., 196, 901-917 (1987)), theloop equivalent to CDR-H1 extends from residue 26 to residue 32. Thus‘CDR-H1’, as used herein, comprises residues 26 to 35, as described by acombination of the Kabat numbering system and Chothia's topological loopdefinition.

The CDRs of the light chain variable domain are located at residues24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3)according to the Kabat numbering system.

In one embodiment the present invention provides a neutralising antibodyhaving specificity for human IL-17A and human IL-17F, comprising a heavychain, wherein the variable domain of the heavy chain comprises at leastone of a CDR having the sequence given in SEQ ID NO:1 for CDR-H1, a CDRhaving the sequence given in SEQ ID NO:2 for CDR-H2 and a CDR having thesequence given in SEQ ID NO:3 for CDR-H3.

In another embodiment the present invention provides a neutralisingantibody having specificity for human IL-17A and human IL-17F,comprising a heavy chain, wherein at least two of CDR-H1, CDR-H2 andCDR-H3 of the variable domain of the heavy chain are selected from thefollowing: the sequence given in SEQ ID NO:1 for CDR-H1, the sequencegiven in SEQ ID NO:2 for CDR-H2 and the sequence given in SEQ ID NO:3for CDR-H3. For example, the antibody may comprise a heavy chain whereinCDR-H1 has the sequence given in SEQ ID NO:1 and CDR-H2 has the sequencegiven in SEQ ID NO:2. Alternatively, the antibody may comprise a heavychain wherein CDR-H1 has the sequence given in SEQ ID NO:1 and CDR-H3has the sequence given in SEQ ID NO:3, or the antibody may comprise aheavy chain wherein CDR-H2 has the sequence given in SEQ ID NO:2 andCDR-H3 has the sequence given in SEQ ID NO:3. For the avoidance ofdoubt, it is understood that all permutations are included.

In another embodiment the present invention provides a neutralisingantibody having specificity for human IL-17A and human IL-17F,comprising a heavy chain, wherein the variable domain of the heavy chaincomprises the sequence given in SEQ ID NO:1 for CDR-H1, the sequencegiven in SEQ ID NO:2 for CDR-H2 and the sequence given in SEQ ID NO:3for CDR-H3.

In one embodiment the present invention provides a neutralising antibodyhaving specificity for human IL-17A and human IL-17F, comprising a lightchain, wherein the variable domain of the light chain comprises at leastone of a CDR having the sequence given in SEQ ID NO:4 for CDR-L1, a CDRhaving the sequence given in SEQ ID NO:5 for CDR-L2 and a CDR having thesequence given in SEQ ID NO:6 for CDR-L3.

In another embodiment the present invention provides a neutralisingantibody having specificity for human IL-17A and human IL-17F,comprising a light chain, wherein at least two of CDR-L1, CDR-L2 andCDR-L3 of the variable domain of the light chain are selected from thefollowing: the sequence given in SEQ ID NO:4 for CDR-L1, the sequencegiven in SEQ ID NO:5 for CDR-L2 and the sequence given in SEQ ID NO:6for CDR-L3. For example, the antibody may comprise a light chain whereinCDR-L1 has the sequence given in SEQ ID NO:4 and CDR-L2 has the sequencegiven in SEQ ID NO:5. Alternatively, the antibody may comprise a lightchain wherein CDR-L1 has the sequence given in SEQ ID NO:4 and CDR-L3has the sequence given in SEQ ID NO:6, or the antibody may comprise alight chain wherein CDR-L2 has the sequence given in SEQ ID NO:5 andCDR-L3 has the sequence given in SEQ ID NO:6. For the avoidance ofdoubt, it is understood that all permutations are included.

In another embodiment the present invention provides a neutralisingantibody having specificity for human IL-17A and human IL-17F,comprising a light chain, wherein the variable domain comprises thesequence given in SEQ ID NO:4 for CDR-L1, the sequence given in SEQ IDNO:5 for CDR-L2 and the sequence given in SEQ ID NO:6 for CDR-L3.

The antibody molecules of the present invention preferably comprise acomplementary light chain or a complementary heavy chain, respectively.

Hence in one embodiment, an antibody according to the present inventioncomprises a heavy chain, wherein the variable domain of the heavy chaincomprises the sequence given in SEQ ID NO:1 for CDR-H1, the sequencegiven in SEQ ID NO:2 for CDR-H2 and the sequence given in SEQ ID NO:3for CDR-H3 and a light chain wherein the variable domain of the lightchain comprises the sequence given in SEQ ID NO:4 for CDR-L1, thesequence given in SEQ ID NO:5 for CDR-L2 and the sequence given in SEQID NO:6 for CDR-L3.

It will be appreciated that one or more amino acid substitutions may bemade to the CDRs provided by the present invention without significantlyaltering the ability of the antibody to bind to IL-17A and IL-17F and toneutralise IL-17A and IL-17F activity. The effect of any amino acidsubstitutions on binding and neutralisation can be readily tested by oneskilled in the art, for example by using the methods described herein.Accordingly, the present invention provides an antibody comprising oneor more CDRs selected from CDRH-1 (SEQ ID NO:1), CDRH-2 (SEQ ID NO:2),CDRH-3 (SEQ ID NO:3), CDRL-1 (SEQ ID NO:4), CDRL-2 (SEQ ID NO:5) andCDRL-3 (SEQ ID NO:6) in which one or more amino acids in one or more ofthe CDRs has been substituted with another amino acid. It will also beappreciated that the length of one or more of the CDRs may be alteredwithout significantly altering the ability of the antibody to bind toIL-17A and IL-17F and to neutralise IL-17A and IL-17F activity.

In one embodiment, an antibody of the present invention comprises aheavy chain, wherein the variable domain of the heavy chain comprisesthree CDRs wherein the sequence of CDRH-1 has at least 60% identity orsimilarity to the sequence given in SEQ ID NO:1, CDRH-2 has at least 60%identity or similarity to the sequence given in SEQ ID NO:2 and/orCDRH-3 has at least 60% identity or similarity to the sequence given inSEQ ID NO:3. In another embodiment, an antibody of the present inventioncomprises a heavy chain, wherein the variable domain of the heavy chaincomprises three CDRs wherein the sequence of CDRH-1 has at least 70%,80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQID NO:1, CDRH-2 has at least 70%, 80%, 90%, 95% or 98% identity orsimilarity to the sequence given in SEQ ID NO:2 and/or CDRH-3 has atleast 70%, 80%, 90%, 95% or 98% identity or similarity to the sequencegiven in SEQ ID NO:3.

“Identity”, as used herein, indicates that at any particular position inthe aligned sequences, the amino acid residue is identical between thesequences. “Similarity”, as used herein, indicates that, at anyparticular position in the aligned sequences, the amino acid residue isof a similar type between the sequences. For example, leucine may besubstituted for isoleucine or valine. Other amino acids which can oftenbe substituted for one another include but are not limited to:

-   -   phenylalanine, tyrosine and tryptophan (amino acids having        aromatic side chains);    -   lysine, arginine and histidine (amino acids having basic side        chains);    -   aspartate and glutamate (amino acids having acidic side chains);    -   asparagine and glutamine (amino acids having amide side chains);        and    -   cysteine and methionine (amino acids having sulphur-containing        side chains). Degrees of identity and similarity can be readily        calculated (Computational Molecular Biology, Lesk, A. M., ed.,        Oxford University Press, New York, 1988; Biocomputing.        Informatics and Genome Projects, Smith, D. W., ed., Academic        Press, New York, 1993; Computer Analysis of Sequence Data, Part        1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New        Jersey, 1994; Sequence Analysis in Molecular Biology, von        Heinje, G., Academic Press, 1987; and Sequence Analysis Primer,        Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,        1991).

In another embodiment, an antibody of the present invention comprises alight chain, wherein the variable domain of the light chain comprisesthree CDRs wherein the sequence of CDRL-1 has at least 60% identity orsimilarity to the sequence given in SEQ ID NO:4, CDRL-2 has at least 60%identity or similarity to the sequence given in SEQ ID NO:5 and/orCDRL-3 has at least 60% identity or similarity to the sequence given inSEQ ID NO:6. In another embodiment, an antibody of the present inventioncomprises a light chain, wherein the variable domain of the heavy chaincomprises three CDRs wherein the sequence of CDRL-1 has at least 70%,80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQID NO:4, CDRL-2 has at least 70%, 80%, 90%, 95% or 98% identity orsimilarity to the sequence given in SEQ ID NO:5 and/or CDRL-3 has atleast 70%, 80%, 90%, 95% or 98% identity or similarity to the sequencegiven in SEQ ID NO:6.

In one embodiment the antibody provided by the present invention is amonoclonal antibody.

In one embodiment the antibody provided by the present invention is achimeric antibody.

In one embodiment the antibody provided by the present invention is aCDR-grafted antibody molecule comprising one or more of the CDRsprovided in SEQ ID NOS:1 to 6. As used herein, the term ‘CDR-graftedantibody molecule’ refers to an antibody molecule wherein the heavyand/or light chain contains one or more CDRs (including, if desired, oneor more modified CDRs) from a donor antibody (e.g. a murine monoclonalantibody) grafted into a heavy and/or light chain variable regionframework of an acceptor antibody (e.g. a human antibody). For a review,see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998. In oneembodiment rather than the entire CDR being transferred, only one ormore of the specificity determining residues from any one of the CDRsdescribed herein above are transferred to the human antibody framework(see for example, Kashmiri et al., 2005, Methods, 36, 25-34). In oneembodiment only the specificity determining residues from one or more ofthe CDRs described herein above are transferred to the human antibodyframework. In another embodiment only the specificity determiningresidues from each of the CDRs described herein above are transferred tothe human antibody framework.

When the CDRs or specificity determining residues are grafted, anyappropriate acceptor variable region framework sequence may be usedhaving regard to the class/type of the donor antibody from which theCDRs are derived, including mouse, primate and human framework regions.Preferably, the CDR-grafted antibody according to the present inventionhas a variable domain comprising human acceptor framework regions aswell as one or more of the CDRs or specificity determining residuesdescribed above. Thus, provided in one embodiment is a neutralisingCDR-grafted antibody wherein the variable domain comprises humanacceptor framework regions and non-human donor CDRs.

Examples of human frameworks which can be used in the present inventionare KOL, NEWM, REI, EU, TUR, TEL LAY and POM (Kabat et al., supra). Forexample, KOL and NEWM can be used for the heavy chain, REI can be usedfor the light chain and EU, LAY and POM can be used for both the heavychain and the light chain. Alternatively, human germline sequences maybe used; these are available at: http://vbase.mrc-cpe.cam.ac.uk/

In a CDR-grafted antibody of the present invention, the acceptor heavyand light chains do not necessarily need to be derived from the sameantibody and may, if desired, comprise composite chains having frameworkregions derived from different chains.

The preferred framework region for the heavy chain of the CDR-graftedantibody of the present invention is derived from the human sub-groupVH3 sequence 1-3 3-07 together with JH4. Accordingly, provided is aneutralising CDR-grafted antibody comprising at least one non-humandonor CDR wherein the heavy chain framework region is derived from thehuman subgroup sequence 1-3 3-07 together with JH4. The sequence ofhuman JH4 is as follows: (YFDY)WGQGTLVTVSS. The YFDY motif is part ofCDR-H3 and is not part of framework 4 (Ravetch, J V. et al., 1981, Cell,27, 583-591).

The preferred framework region for the light chain of the CDR-graftedantibody of the present invention is derived from the human germlinesub-group VK1 sequence 2-1-(1) L4 together with JK1. Accordingly,provided is a neutralising CDR-grafted antibody comprising at least onenon-human donor CDR wherein the light chain framework region is derivedfrom the human subgroup sequence VK1 2-1-(1) L4 together with JK1. TheJK1 sequence is as follows: (WT)FGQGTKVEIK. The WT motif is part ofCDR-L3 and is not part of framework 4 (Hieter, P A., et al., 1982, J.Biol. Chem., 257, 1516-1522).

Also, in a CDR-grafted antibody of the present invention, the frameworkregions need not have exactly the same sequence as those of the acceptorantibody. For instance, unusual residues may be changed to morefrequently-occurring residues for that acceptor chain class or type.Alternatively, selected residues in the acceptor framework regions maybe changed so that they correspond to the residue found at the sameposition in the donor antibody (see Reichmann et al., 1998, Nature, 332,323-324). Such changes should be kept to the minimum necessary torecover the affinity of the donor antibody. A protocol for selectingresidues in the acceptor framework regions which may need to be changedis set forth in WO 91/09967.

Preferably, in a CDR-grafted antibody molecule of the present invention,if the acceptor heavy chain has the human VH3 sequence 1-3 3-07 togetherwith JH4, then the acceptor framework regions of the heavy chaincomprise, in addition to one or more donor CDRs, a donor residue at atleast position 94 (according to Kabat et al., (supra)). Accordingly,provided is a CDR-grafted antibody, wherein at least the residue atposition 94 of the variable domain of the heavy chain is a donorresidue.

Preferably, in a CDR-grafted antibody molecule according to the presentinvention, if the acceptor light chain has the human sub-group VK1sequence 2-1-(1) L4 together with JK1, then no donor residues aretransferred i.e. only the CDRs are transferred. Accordingly, provided isa CDR-grafted antibody wherein only the CDRs are transferred to thedonor framework.

Donor residues are residues from the donor antibody, i.e. the antibodyfrom which the CDRs were originally derived.

In one embodiment, an antibody of the present invention comprises aheavy chain, wherein the variable domain of the heavy chain comprisesthe sequence given in SEQ ID NO:9 (gH9).

In another embodiment, an antibody of the present invention comprises aheavy chain, wherein the variable domain of the heavy chain comprises asequence having at least 60% identity or similarity to the sequencegiven in SEQ ID NO:9. In one embodiment, an antibody of the presentinvention comprises a heavy chain, wherein the variable domain of theheavy chain comprises a sequence having at least 70%, 80%, 90%, 95% or98% identity or similarity to the sequence given in SEQ ID NO:9.

In one embodiment, an antibody of the present invention comprises alight chain, wherein the variable domain of the light chain comprisesthe sequence given in SEQ ID NO:7 (gL7).

In another embodiment, an antibody of the present invention comprises alight chain, wherein the variable domain of the light chain comprises asequence having at least 60% identity or similarity to the sequencegiven in SEQ ID NO:7. In one embodiment the antibody of the presentinvention comprises a light chain, wherein the variable domain of thelight chain comprises a sequence having at least 70%, 80%, 90%, 95% or98% identity or similarity to the sequence given in SEQ ID NO:7.

In one embodiment an antibody of the present invention comprises a heavychain, wherein the variable domain of the heavy chain comprises thesequence given in SEQ ID NO:9 and a light chain, wherein the variabledomain of the light chain comprises the sequence given in SEQ ID NO:7.

In another embodiment of the invention, the antibody comprises a heavychain and a light chain, wherein the variable domain of the heavy chaincomprises a sequence having at least 60% identity or similarity to thesequence given in SEQ ID NO:9 and the variable domain of the light chaincomprises a sequence having at least 60% identity or similarity to thesequence given in SEQ ID NO:7. Preferably, the antibody comprises aheavy chain, wherein the variable domain of the heavy chain comprises asequence having at least 70%, 80%, 90%, 95% or 98% identity orsimilarity to the sequence given in SEQ ID NO:9 and a light chain,wherein the variable domain of the light chain comprises a sequencehaving at least 70%, 80%, 90%, 95% or 98% identity or similarity to thesequence given in SEQ ID NO:7.

As described herein above, the antibody molecule of the presentinvention may comprise a complete antibody molecule having full lengthheavy and light chains or a fragment thereof, such as a domain antibodye.g. VH, VL, VHH, Fab, modified Fab, Fab′, F(ab′)₂, Fv or scFv fragment.

The constant region domains of the antibody molecule of the presentinvention, if present, may be selected having regard to the proposedfunction of the antibody molecule, and in particular the effectorfunctions which may be required. For example, the constant regiondomains may be human IgA, IgD, IgE, IgG or IgM domains. In particular,human IgG constant region domains may be used, especially of the IgG1and IgG3 isotypes when the antibody molecule is intended for therapeuticuses and antibody effector functions are required. Alternatively, IgG2and IgG4 isotypes may be used when the antibody molecule is intended fortherapeutic purposes and antibody effector functions are not required,e.g. for simply blocking IL-17 activity. For example IgG4 molecules inwhich the serine at position 241 has been changed to proline asdescribed in Angal et al., Molecular Immunology, 1993, 30 (1), 105-108may be used. Particularly preferred is the IgG4 constant domaincomprising this change.

In one embodiment the antibody heavy chain comprises a CH1 domain andthe antibody light chain comprises a CL domain, either kappa or lambda.

In a preferred embodiment the antibody provided by the present inventionis a neutralising antibody having specificity for human IL-17A and humanIL-17F in which the heavy chain constant region comprises the human IgG4constant region in which the serine at position 241 has been substitutedby proline as described in Angal et al., supra. Accordingly, the presentinvention provides an antibody in which the heavy chain comprises orconsists of the sequence given in SEQ ID NO:15.

In one embodiment of the invention, the antibody comprises a heavychain, wherein the heavy chain comprises a sequence having at least 60%identity or similarity to the sequence given in SEQ ID NO:15.Preferably, the antibody comprises a heavy chain, wherein the heavychain comprises a sequence having at least 70%, 80%, 90%, 95% or 98%identity or similarity to the sequence given in SEQ ID NO:15.

In one embodiment an antibody molecule according to the presentinvention comprises a light chain comprising the sequence given in SEQID NO:11.

In one embodiment of the invention, the antibody comprises a lightchain, wherein the light chain comprises a sequence having at least 60%identity or similarity to the sequence given in SEQ ID NO:11.Preferably, the antibody comprises a light chain, wherein the lightchain comprises a sequence having at least 70%, 80%, 90%, 95% or 98%identity or similarity to the sequence given in SEQ ID NO:11.

In one embodiment the present invention provides an antibody in whichthe heavy chain comprises or consists of the sequence given in SEQ IDNO:15 and the light chain comprises or consists of the sequence given inSEQ ID NO:11.

In one embodiment of the invention, the antibody comprises a heavy chainand a light chain, wherein the heavy chain comprises a sequence havingat least 60% identity or similarity to the sequence given in SEQ IDNO:15 and the light chain comprises a sequence having at least 60%identity or similarity to the sequence given in SEQ ID NO:11.Preferably, the antibody comprises a heavy chain, wherein the heavychain comprises a sequence having at least 70%, 80%, 90%, 95% or 98%identity or similarity to the sequence given in SEQ ID NO:15 and a lightchain, wherein the light chain comprises a sequence having at least 70%,80%, 90%, 95% or 98% identity or similarity to the sequence given in SEQID NO:11.

Also provided by the present invention is a specific region or epitopeof human IL-17A and/or a specific region or epitope of human IL-17Fand/or a specific region or epitope of human IL-17A/F heterodimer whichis bound by an antibody provided by the present invention, in particularan antibody comprising the heavy chain sequence gH9 (SEQ ID NO:9) and/orthe light chain sequence gL7 (SEQ ID NO:7).

The specific region or epitope of the human IL-17A polypeptide and thespecific region or epitope of the human IL-17F polypeptide and thespecific region or epitope of the human IL-17A/F heterodimer can beidentified by any suitable epitope mapping method known in the art incombination with any one of the antibodies provided by the presentinvention. Examples of such methods include screening peptides ofvarying lengths derived from IL-17A and IL-17F for binding to theantibody of the present invention with the smallest fragment that canspecifically bind to the antibody containing the sequence of the epitoperecognised by the antibody. The IL-17 peptides may be producedsynthetically or by proteolytic digestion of the appropriate IL-17polypeptide. Peptides that bind the antibody can be identified by, forexample, mass spectrometric analysis. In another example, NMRspectroscopy can be used to identify the epitope bound by an antibody ofthe present invention. Once identified, the epitopic fragment whichbinds an antibody of the present invention can be used, if required, asan immunogen to obtain additional neutralising antibodies which bind thesame epitope.

Antibodies which cross-block the binding of an antibody according to thepresent invention, in particular, an antibody comprising the heavy chainsequence gH9 (SEQ ID NO:9) and the light chain sequence gL7 (SEQ IDNO:7), may be similarly useful in neutralising IL-17A and IL-17Factivity. Accordingly, the present invention also provides aneutralising antibody which binds human IL-17A and human IL-17F, whichcross-blocks the binding of any one of the antibodies described above tohuman IL-17A and/or human IL-17F and/or human IL-17A/F heterodimerand/or is cross-blocked from binding IL-17A and/or IL-17F and/or humanIL-17A/F heterodimer by any one of those antibodies. In one embodiment,such an antibody binds to the same epitope as an antibody describedherein above. In another embodiment the cross-blocking neutralisingantibody binds to an epitope which borders and/or overlaps with theepitope bound by an antibody described herein above. In anotherembodiment the cross-blocking neutralising antibody of this aspect ofthe invention does not bind to the same epitope as an antibody of thepresent invention or an epitope that borders and/or overlaps with saidepitope.

Cross-blocking antibodies can be identified using any suitable method inthe art, for example by using competition ELISA or BIAcore where bindingof the cross blocking antibody to human IL-17A and/or human IL-17Fprevents the binding of an antibody of the present invention or viceversa.

In one embodiment there is provided a neutralising antibody which bindsto human IL-17A and human IL-17F, which cross-blocks the binding of anantibody whose heavy chain comprises the sequence gH9 (SEQ ID NO:9) andwhose light chain comprises the sequence gL7 (SEQ ID NO:7) to humanIL-17A and to human IL-17F. In one embodiment the cross-blockingantibodies provided by the present invention inhibit the binding of anantibody comprising the heavy chain sequence gH9 (SEQ ID NO:9) and thelight chain sequence gL7 (SEQ ID NO:7) to IL-17A by greater than 80%,preferably by greater than 85%, more preferably by greater than 90%,even more preferably by greater than 95% and to IL-17F by greater than80%, preferably by greater than 85%, more preferably by greater than90%, even more preferably by greater than 95%.

In one embodiment there is provided a neutralising antibody which bindsto human IL-17A and human IL-17F, which cross-blocks the binding of anantibody whose heavy chain comprises the sequence gH9 (SEQ ID NO:9) andwhose light chain comprises the sequence gL7 (SEQ ID NO:7) to humanIL-17A and to human IL-17F and to human IL-17A/F heterodimer. In oneembodiment the cross-blocking antibodies provided by the presentinvention inhibit the binding of an antibody comprising the heavy chainsequence gH9 (SEQ ID NO:9) and the light chain sequence gL7 (SEQ IDNO:7) to IL-17A by greater than 80%, preferably by greater than 85%,more preferably by greater than 90%, even more preferably by greaterthan 95% and to IL-17F by greater than 80%, preferably by greater than85%, more preferably by greater than 90%, even more preferably bygreater than 95% and to IL-17A/F heterodimer to IL-17F by greater than80%, preferably by greater than 85%, more preferably by greater than90%, even more preferably by greater than 95%.

In one embodiment there is provided a neutralising antibody which bindsto human IL-17A and human IL-17F, which cross-blocks the binding of anantibody whose heavy chain comprises the sequence gH9 (SEQ ID NO:9) andwhose light chain comprises the sequence gL7 (SEQ ID NO:7) to humanIL-17A or to human IL-17F or human IL-17A/F heterodimer. In oneembodiment the cross-blocking antibodies provided by the presentinvention inhibit the binding of an antibody comprising the heavy chainsequence gH9 (SEQ ID NO:9) and the light chain sequence gL7 (SEQ IDNO:7) to IL-17A or IL-17F or IL-17A/F by greater than 80%, preferably bygreater than 85%, more preferably by greater than 90%, even morepreferably by greater than 95%.

Alternatively or in addition, neutralising antibodies according to thisaspect of the invention may be cross-blocked from binding to humanIL-17A and human IL-17F by an antibody comprising the heavy chainsequence gH9 (SEQ ID NO:9) and the light chain sequence gL7 (SEQ IDNO:7). Also provided therefore is a neutralising antibody molecule whichbinds to human IL-17A and to human IL-17F which is cross-blocked frombinding human IL-17A and human IL-17F by an antibody comprising theheavy chain sequence gH9 (SEQ ID NO:9) and the light chain sequence gL7(SEQ ID NO:7). In one embodiment the neutralising antibodies provided bythis aspect of the invention are inhibited from binding to human IL-17Aand human IL-17F by an antibody comprising the heavy chain sequence gH9(SEQ ID NO:9) and the light chain sequence gL7 (SEQ ID NO:7) by greaterthan 80%, preferably by greater than 85%, more preferably by greaterthan 90%, even more preferably by greater than 95%.

In another embodiment there is provided a neutralising antibody moleculewhich binds to human IL-17A and to human IL-17F which is cross-blockedfrom binding human IL-17A and human IL-17F and IL-17A/F heterodimer byan antibody comprising the heavy chain sequence gH9 (SEQ ID NO:9) andthe light chain sequence gL7 (SEQ ID NO:7). In one embodiment theneutralising antibodies provided by this aspect of the invention areinhibited from binding to human IL-17A and human IL-17F and humanIL-17A/F heterodimer by an antibody comprising the heavy chain sequencegH9 (SEQ ID NO:9) and the light chain sequence gL7 (SEQ ID NO:7) bygreater than 80%, preferably by greater than 85%, more preferably bygreater than 90%, even more preferably by greater than 95%.

Also provided therefore is a neutralising antibody molecule which bindsto human IL-17A and to human IL-17F which is cross-blocked from bindinghuman IL-17A or human IL-17F or human IL-17A/F by an antibody comprisingthe heavy chain sequence gH9 (SEQ ID NO:9) and the light chain sequencegL7 (SEQ ID NO:7). In one embodiment the neutralising antibodiesprovided by this aspect of the invention are inhibited from binding tohuman IL-17A or human IL-17F or human IL-17A/F by an antibody comprisingthe heavy chain sequence gH9 (SEQ ID NO:9) and the light chain sequencegL7 (SEQ ID NO:7) by greater than 80%, preferably by greater than 85%,more preferably by greater than 90%, even more preferably by greaterthan 95%.

The antibody molecule of any aspect of the present invention preferablyhas a high binding affinity, preferably nanomolar, even more preferablypicomolar. It will be appreciated that the binding affinity of anantibody according to the present invention for human IL-17A may bedifferent from the binding affinity of the same antibody for humanIL-17F and/or the IL-17A/F heterodimer. In one example the antibodymolecule of the present invention has an affinity for IL-17A that isgreater than its affinity for IL-17F. In one example the antibodymolecule of the present invention has an affinity for IL-17A which is atleast 10 fold greater than its binding affinity for IL-17F. In oneexample the antibody molecule of the present invention has an affinityfor IL-17A which is at least 50 fold greater than its binding affinityfor IL-17F. In one example the antibody molecule of the presentinvention has an affinity for IL-17A which is at least 100 fold greaterthan its binding affinity for IL-17F. In one example the antibodymolecule of the present invention has an affinity for IL-17F that isgreater than its affinity for IL-17A. In one example the antibodymolecule of the present invention has an affinity for IL-17A that is thesame as its affinity for IL-17F. In one example the antibody molecule ofthe present invention has a picomolar affinity for IL-17A and ananomolar affinity for IL-17F. In one example the antibody molecule ofthe present invention has a nanomolar affinity for IL-17F and apicomolar affinity for IL-17A. In one example the antibody molecule ofthe present invention has a nanomolar affinity for both IL-17A andIL-17F. In one example the antibody molecule of the present inventionhas a picomolar affinity for both IL-17A and IL-17F.

Preferably the antibody molecule of the present invention has a bindingaffinity for IL-17A of better than 10 nM. In one embodiment the antibodymolecule of the present invention has a binding affinity for IL-17A ofbetter than 500 pM. In one embodiment the antibody molecule of thepresent invention has a binding affinity for IL-17A of better than 100pM. In one embodiment the antibody molecule of the present invention hasa binding affinity for IL-17A of better than 20 pM. In one embodimentthe antibody of the present invention has an affinity for IL-17A of 16pM.

Preferably the antibody molecule of the present invention has a bindingaffinity for IL-17F of better than 10 nM. In one embodiment the antibodyof the present invention has an affinity for IL-17F of better than 2 nM.In one embodiment the antibody of the present invention has an affinityfor IL-17F of 1.75 nM.

Preferably the antibody molecule of the present invention has a bindingaffinity for IL-17A/F heterodimer of better than 10 nM. In oneembodiment the antibody molecule of the present invention has a bindingaffinity for IL-17A/F heterodimer of better than 500 pM. In oneembodiment the antibody molecule of the present invention has a bindingaffinity for IL-17A/F heterodimer of better than 150 pM. In oneembodiment the antibody molecule of the present invention has a bindingaffinity for IL-17A/F heterodimer of 116 pM.

In one embodiment the antibody molecule of the present invention has abinding affinity for cynomolgus IL-17F of better than 2 nM. In oneembodiment the antibody molecule of the present invention has a bindingaffinity for cynomolgus IL-17F of 1.03 nM.

It will be appreciated that the affinity of antibodies provided by thepresent invention may be altered using any suitable method known in theart. The present invention therefore also relates to variants of theantibody molecules of the present invention, which have an improvedaffinity for IL-17A and/or IL-17F. Such variants can be obtained by anumber of affinity maturation protocols including mutating the CDRs(Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Markset al., Bio/Technology, 10, 779-783, 1992), use of mutator strains of E.coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling(Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phagedisplay (Thompson et al., J. Mol. Biol., 256, 77-88, 1996) and sexualPCR (Crameri et al., Nature, 391, 288-291, 1998). Vaughan et al. (supra)discusses these methods of affinity maturation.

In one embodiment the antibody molecules of the present inventionneutralise IL-17A and IL-17F activity, for example in the in vitroassays described in the Examples. In one embodiment the presentinvention provides a neutralising antibody having specificity for humanIL-17A and IL-17F which is capable of inhibiting the activity of 0.8 nMhuman IL-17A by 50% at a concentration of less than 5 nM and theactivity of 4.2 nM IL-17F by 50% at a concentration of less than 12 nMsaid inhibitory activity being measured on the IL-17A or IL-17F inducedrelease of IL-6 from Hela cells. In one embodiment the concentration ofantibody which inhibits IL-17A by 50% is less than 3 nM. In oneembodiment the concentration of antibody which inhibits IL-17F by 50% isless than 11 nM. In one embodiment the human IL-17A and human IL-17Fused in the assay are recombinant human IL-17A and IL-17F. In oneembodiment the neutralising antibody is a humanised or fully humanantibody.

If desired an antibody for use in the present invention may beconjugated to one or more effector molecule(s). It will be appreciatedthat the effector molecule may comprise a single effector molecule ortwo or more such molecules so linked as to form a single moiety that canbe attached to the antibodies of the present invention. Where it isdesired to obtain an antibody fragment linked to an effector molecule,this may be prepared by standard chemical or recombinant DNA proceduresin which the antibody fragment is linked either directly or via acoupling agent to the effector molecule. Techniques for conjugating sucheffector molecules to antibodies are well known in the art (see,Hellstrom et al., Controlled Drug Delivery, 2nd Ed., Robinson et al.,eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123).Particular chemical procedures include, for example, those described inWO 93/06231, WO 92/22583, WO 89/00195, WO 89/01476 and WO03031581.Alternatively, where the effector molecule is a protein or polypeptidethe linkage may be achieved using recombinant DNA procedures, forexample as described in WO 86/01533 and EP0392745.

The term effector molecule as used herein includes, for example,antineoplastic agents, drugs, toxins, biologically active proteins, forexample enzymes, other antibody or antibody fragments, synthetic ornaturally occurring polymers, nucleic acids and fragments thereof e.g.DNA, RNA and fragments thereof, radionuclides, particularly radioiodide,radioisotopes, chelated metals, nanoparticles and reporter groups suchas fluorescent compounds or compounds which may be detected by NMR orESR spectroscopy.

Examples of effector molecules may include cytotoxins or cytotoxicagents including any agent that is detrimental to (e.g. kills) cells.Examples include combrestatins, dolastatins, epothilones, staurosporin,maytansinoids, spongistatins, rhizoxin, halichondrins, roridins,hemiasterlins, taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof.

Effector molecules also include, but are not limited to, antimetabolites(e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g. daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g. dactinomycin (formerly actinomycin),bleomycin, mithramycin, anthramycin (AMC), calicheamicins orduocarmycins), and anti-mitotic agents (e.g. vincristine andvinblastine).

Other effector molecules may include chelated radionuclides such as¹¹¹In and ⁹⁰Y, Lu¹⁷⁷, Bismuth²¹³, Californium²⁵², Iridium¹⁹² andTungsten¹⁸⁸/Rhenium¹⁸⁸; or drugs such as but not limited to,alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

Other effector molecules include proteins, peptides and enzymes. Enzymesof interest include, but are not limited to, proteolytic enzymes,hydrolases, lyases, isomerases, transferases. Proteins, polypeptides andpeptides of interest include, but are not limited to, immunoglobulins,toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheriatoxin, a protein such as insulin, tumour necrosis factor, α-interferon,β-interferon, nerve growth factor, platelet derived growth factor ortissue plasminogen activator, a thrombotic agent or an anti-angiogenicagent, e.g. angiostatin or endostatin, or, a biological responsemodifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2(IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (G-CSF), nervegrowth factor (NGF) or other growth factor and immunoglobulins.

Other effector molecules may include detectable substances useful forexample in diagnosis. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive nuclides, positronemitting metals (for use in positron emission tomography), andnonradioactive paramagnetic metal ions. See generally U.S. Pat. No.4,741,900 for metal ions which can be conjugated to antibodies for useas diagnostics. Suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;suitable prosthetic groups include streptavidin, avidin and biotin;suitable fluorescent materials include umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride and phycoerythrin; suitable luminescentmaterials include luminol; suitable bioluminescent materials includeluciferase, luciferin, and aequorin; and suitable radioactive nuclidesinclude ¹²⁵I, ¹³¹I, ¹¹¹In and ⁹⁹Tc.

In another example the effector molecule may increase the half-life ofthe antibody in vivo, and/or reduce immunogenicity of the antibodyand/or enhance the delivery of an antibody across an epithelial barrierto the immune system. Examples of suitable effector molecules of thistype include polymers, albumin, albumin binding proteins or albuminbinding compounds such as those described in WO05/117984.

Where the effector molecule is a polymer it may, in general, be asynthetic or a naturally occurring polymer, for example an optionallysubstituted straight or branched chain polyalkylene, polyalkenylene orpolyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g.a homo- or hetero-polysaccharide.

Particular optional substituents which may be present on theabove-mentioned synthetic polymers include one or more hydroxy, methylor methoxy groups.

Particular examples of synthetic polymers include optionally substitutedstraight or branched chain poly(ethyleneglycol), poly(propyleneglycol)poly(vinylalcohol) or derivatives thereof, especially optionallysubstituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol) orderivatives thereof.

Particular naturally occurring polymers include lactose, amylose,dextran, glycogen or derivatives thereof.

“Derivatives” as used herein is intended to include reactivederivatives, for example thiol-selective reactive groups such asmaleimides and the like. The reactive group may be linked directly orthrough a linker segment to the polymer. It will be appreciated that theresidue of such a group will in some instances form part of the productas the linking group between the antibody fragment and the polymer.

The size of the polymer may be varied as desired, but will generally bein an average molecular weight range from 500 Da to 50000 Da, preferablyfrom 5000 to 40000 Da and more preferably from 20000 to 40000 Da. Thepolymer size may in particular be selected on the basis of the intendeduse of the product for example ability to localize to certain tissuessuch as tumors or extend circulating half-life (for review see Chapman,2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example,where the product is intended to leave the circulation and penetratetissue, for example for use in the treatment of a tumour, it may beadvantageous to use a small molecular weight polymer, for example with amolecular weight of around 5000 Da. For applications where the productremains in the circulation, it may be advantageous to use a highermolecular weight polymer, for example having a molecular weight in therange from 20000 Da to 40000 Da.

Particularly preferred polymers include a polyalkylene polymer, such asa poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) ora derivative thereof, and especially with a molecular weight in therange from about 15000 Da to about 40000 Da.

In one example antibodies for use in the present invention are attachedto poly(ethyleneglycol) (PEG) moieties. In one particular example theantibody is an antibody fragment and the PEG molecules may be attachedthrough any available amino acid side-chain or terminal amino acidfunctional group located in the antibody fragment, for example any freeamino, imino, thiol, hydroxyl or carboxyl group. Such amino acids mayoccur naturally in the antibody fragment or may be engineered into thefragment using recombinant DNA methods (see for example U.S. Pat. Nos.5,219,996; 5,667,425; WO98/25971). In one example the antibody moleculeof the present invention is a modified Fab fragment wherein themodification is the addition to the C-terminal end of its heavy chainone or more amino acids to allow the attachment of an effector molecule.Preferably, the additional amino acids form a modified hinge regioncontaining one or more cysteine residues to which the effector moleculemay be attached. Multiple sites can be used to attach two or more PEGmolecules.

Preferably PEG molecules are covalently linked through a thiol group ofat least one cysteine residue located in the antibody fragment. Eachpolymer molecule attached to the modified antibody fragment may becovalently linked to the sulphur atom of a cysteine residue located inthe fragment. The covalent linkage will generally be a disulphide bondor, in particular, a sulphur-carbon bond. Where a thiol group is used asthe point of attachment appropriately activated effector molecules, forexample thiol selective derivatives such as maleimides and cysteinederivatives may be used. An activated polymer may be used as thestarting material in the preparation of polymer-modified antibodyfragments as described above. The activated polymer may be any polymercontaining a thiol reactive group such as an α-halocarboxylic acid orester, e.g. iodoacetamide, an imide, e.g. maleimide, a vinyl sulphone ora disulphide. Such starting materials may be obtained commercially (forexample from Nektar, formerly Shearwater Polymers Inc., Huntsville,Ala., USA) or may be prepared from commercially available startingmaterials using conventional chemical procedures. Particular PEGmolecules include 20K methoxy-PEG-amine (obtainable from Nektar,formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA(obtainable from Nektar, formerly Shearwater).

In one embodiment, the antibody is a modified Fab fragment which isPEGylated, i.e. has PEG (poly(ethyleneglycol)) covalently attachedthereto, e.g. according to the method disclosed in EP 0948544 [see also“Poly(ethyleneglycol) Chemistry, Biotechnical and BiomedicalApplications”, 1992, J. Milton Harris (ed), Plenum Press, New York,“Poly(ethyleneglycol) Chemistry and Biological Applications”, 1997, J.Milton Harris and S. Zalipsky (eds), American Chemical Society,Washington D.C. and “Bioconjugation Protein Coupling Techniques for theBiomedical Sciences”, 1998, M. Aslam and A. Dent, Grove Publishers, NewYork; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002,54:531-545]. In one example PEG is attached to a cysteine in the hingeregion. In one example, a PEG modified Fab fragment has a maleimidegroup covalently linked to a single thiol group in a modified hingeregion. A lysine residue may be covalently linked to the maleimide groupand to each of the amine groups on the lysine residue may be attached amethoxypoly(ethyleneglycol) polymer having a molecular weight ofapproximately 20,000 Da. The total molecular weight of the PEG attachedto the Fab fragment may therefore be approximately 40,000 Da.

In one embodiment, the present invention provides a neutralisingantibody molecule having specificity for human IL-17A and human IL-17F,which is a modified Fab fragment having a heavy chain comprising thesequence given in SEQ ID NO:9 and a light chain comprising the sequencegiven in SEQ ID NO:7 and having at the C-terminal end of its heavy chaina modified hinge region containing at least one cysteine residue towhich an effector molecule is attached. Preferably the effector moleculeis PEG and is attached using the methods described in (WO98/25971 andWO2004072116) whereby a lysyl-maleimide group is attached to thecysteine residue at the C-terminal end of the heavy chain, and eachamino group of the lysyl residue has covalently linked to it amethoxypoly(ethyleneglycol) residue having a molecular weight of about20,000 Da. The total molecular weight of the PEG attached to theantibody is therefore approximately 40,000 Da.

In another example effector molecules may be attached to antibodyfragments using the methods described in International patentapplications WO2005/003169, WO2005/003170 and WO2005/003171.

The present invention also provides an isolated DNA sequence encodingthe heavy and/or light chain(s) of an antibody molecule of the presentinvention. Preferably, the DNA sequence encodes the heavy or the lightchain of an antibody molecule of the present invention. The DNA sequenceof the present invention may comprise synthetic DNA, for instanceproduced by chemical processing, cDNA, genomic DNA or any combinationthereof.

DNA sequences which encode an antibody molecule of the present inventioncan be obtained by methods well known to those skilled in the art. Forexample, DNA sequences coding for part or all of the antibody heavy andlight chains may be synthesised as desired from the determined DNAsequences or on the basis of the corresponding amino acid sequences.

DNA coding for acceptor framework sequences is widely available to thoseskilled in the art and can be readily synthesised on the basis of theirknown amino acid sequences.

Standard techniques of molecular biology may be used to prepare DNAsequences coding for the antibody molecule of the present invention.Desired DNA sequences may be synthesised completely or in part usingoligonucleotide synthesis techniques. Site-directed mutagenesis andpolymerase chain reaction (PCR) techniques may be used as appropriate.

Examples of suitable sequences are provided in SEQ ID NO:8; SEQ IDNO:10; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:17 and SEQ ID NO:18.Nucleotides 1-57 in SEQ ID NO 18 and 1-60 in SEQ ID NO 14 encode thesignal peptide sequence from mouse antibody B72.3 (Whittle et al., 1987,Protein Eng. 1 (6) 499-505.) which is cleaved to give a neutralisingantibody molecule of the present invention.

The present invention also relates to a cloning or expression vectorcomprising one or more DNA sequences of the present invention.Accordingly, provided is a cloning or expression vector comprising oneor more DNA sequences encoding an antibody of the present invention.Preferably, the cloning or expression vector comprises two DNAsequences, encoding the light chain and the heavy chain of the antibodymolecule of the present invention, respectively. Preferably, a vectoraccording to the present invention comprises the sequences given in SEQID NO:14 and SEQ ID NO:18. Nucleotides 1-57 in SEQ ID NO 18 and 1-60 inSEQ ID NO 14 encode the signal peptide sequence from mouse antibodyB72.3 (residues 1-19 in SEQ ID NO: 16 and 1-20 in SEQ ID NO:12respectively) which is most preferably cleaved to give a neutralisingantibody molecule of the present invention.

General methods by which the vectors may be constructed, transfectionmethods and culture methods are well known to those skilled in the art.In this respect, reference is made to “Current Protocols in MolecularBiology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and theManiatis Manual produced by Cold Spring Harbor Publishing.

Also provided is a host cell comprising one or more cloning orexpression vectors comprising one or more DNA sequences encoding anantibody of the present invention. Any suitable host cell/vector systemmay be used for expression of the DNA sequences encoding the antibodymolecule of the present invention. Bacterial, for example E. coli, andother microbial systems may be used or eukaryotic, for examplemammalian, host cell expression systems may also be used. Suitablemammalian host cells include CHO, myeloma or hybridoma cells.

The present invention also provides a process for the production of anantibody molecule according to the present invention comprisingculturing a host cell containing a vector of the present invention underconditions suitable for leading to expression of protein from DNAencoding the antibody molecule of the present invention, and isolatingthe antibody molecule.

The antibody molecule may comprise only a heavy or light chainpolypeptide, in which case only a heavy chain or light chain polypeptidecoding sequence needs to be used to transfect the host cells. Forproduction of products comprising both heavy and light chains, the cellline may be transfected with two vectors, a first vector encoding alight chain polypeptide and a second vector encoding a heavy chainpolypeptide. Alternatively, a single vector may be used, the vectorincluding sequences encoding light chain and heavy chain polypeptides.

As the antibodies of the present invention are useful in the treatmentand/or prophylaxis of a pathological condition, the present inventionalso provides a pharmaceutical or diagnostic composition comprising anantibody molecule of the present invention in combination with one ormore of a pharmaceutically acceptable excipient, diluent or carrier.Accordingly, provided is the use of an antibody according to the presentinvention for the manufacture of a medicament. The composition willusually be supplied as part of a sterile, pharmaceutical compositionthat will normally include a pharmaceutically acceptable carrier. Apharmaceutical composition of the present invention may additionallycomprise a pharmaceutically-acceptable adjuvant.

The present invention also provides a process for preparation of apharmaceutical or diagnostic composition comprising adding and mixingthe antibody molecule of the present invention together with one or moreof a pharmaceutically acceptable excipient, diluent or carrier.

The antibody molecule may be the sole active ingredient in thepharmaceutical or diagnostic composition or may be accompanied by otheractive ingredients including other antibody ingredients, for exampleanti-TNF, anti-IL-1β, anti-T cell, anti-IFNγ or anti-LPS antibodies, ornon-antibody ingredients such as xanthines.

The pharmaceutical compositions preferably comprise a therapeuticallyeffective amount of the antibody of the invention. The term“therapeutically effective amount” as used herein refers to an amount ofa therapeutic agent needed to treat, ameliorate or prevent a targeteddisease or condition, or to exhibit a detectable therapeutic orpreventative effect. For any antibody, the therapeutically effectiveamount can be estimated initially either in cell culture assays or inanimal models, usually in rodents, rabbits, dogs, pigs or primates. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

The precise therapeutically effective amount for a human subject willdepend upon the severity of the disease state, the general health of thesubject, the age, weight and gender of the subject, diet, time andfrequency of administration, drug combination(s), reaction sensitivitiesand tolerance/response to therapy. This amount can be determined byroutine experimentation and is within the judgement of the clinician.Generally, a therapeutically effective amount will be from 0.01 mg/kg to50 mg/kg, preferably 0.1 mg/kg to 20 mg/kg. Pharmaceutical compositionsmay be conveniently presented in unit dose forms containing apredetermined amount of an active agent of the invention per dose.

Compositions may be administered individually to a patient or may beadministered in combination (e.g. simultaneously, sequentially orseparately) with other agents, drugs or hormones.

The dose at which the antibody molecule of the present invention isadministered depends on the nature of the condition to be treated, theextent of the inflammation present and on whether the antibody moleculeis being used prophylactically or to treat an existing condition.

The frequency of dose will depend on the half-life of the antibodymolecule and the duration of its effect. If the antibody molecule has ashort half-life (e.g. 2 to 10 hours) it may be necessary to give one ormore doses per day. Alternatively, if the antibody molecule has a longhalf life (e.g. 2 to 15 days) it may only be necessary to give a dosageonce per day, once per week or even once every 1 or 2 months.

The pharmaceutically acceptable carrier should not itself induce theproduction of antibodies harmful to the individual receiving thecomposition and should not be toxic. Suitable carriers may be large,slowly metabolised macromolecules such as proteins, polypeptides,liposomes, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers and inactive virusparticles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the patient.

Preferred forms for administration include forms suitable for parenteraladministration, e.g. by injection or infusion, for example by bolusinjection or continuous infusion. Where the product is for injection orinfusion, it may take the form of a suspension, solution or emulsion inan oily or aqueous vehicle and it may contain formulatory agents, suchas suspending, preservative, stabilising and/or dispersing agents.Alternatively, the antibody molecule may be in dry form, forreconstitution before use with an appropriate sterile liquid.

Once formulated, the compositions of the invention can be administereddirectly to the subject. The subjects to be treated can be animals.However, it is preferred that the compositions are adapted foradministration to human subjects.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, transcutaneous (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical,sublingual, intravaginal or rectal routes. Hyposprays may also be usedto administer the pharmaceutical compositions of the invention.Typically, the therapeutic compositions may be prepared as injectables,either as liquid solutions or suspensions. Solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Dosage treatmentmay be a single dose schedule or a multiple dose schedule.

It will be appreciated that the active ingredient in the compositionwill be an antibody molecule. As such, it will be susceptible todegradation in the gastrointestinal tract. Thus, if the composition isto be administered by a route using the gastrointestinal tract, thecomposition will need to contain agents which protect the antibody fromdegradation but which release the antibody once it has been absorbedfrom the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Remington's Pharmaceutical Sciences (Mack PublishingCompany, N.J. 1991).

It is also envisaged that the antibody of the present invention will beadministered by use of gene therapy. In order to achieve this, DNAsequences encoding the heavy and light chains of the antibody moleculeunder the control of appropriate DNA components are introduced into apatient such that the antibody chains are expressed from the DNAsequences and assembled in situ.

The present invention also provides an antibody molecule for use in thecontrol of inflammatory diseases. Preferably, the antibody molecule canbe used to reduce the inflammatory process or to prevent theinflammatory process.

The present invention also provides the antibody molecule of the presentinvention for use in the treatment or prophylaxis of a pathologicaldisorder that is mediated by IL-17A and/or IL-17F or is associated withan increased level of IL-17A and/or IL-17F. Preferably, the pathologicalcondition is selected from the group consisting of infections (viral,bacterial, fungal and parasitic), endotoxic shock associated withinfection, arthritis, rheumatoid arthritis, asthma, pelvic inflammatorydisease, Alzheimer's Disease, Crohn's disease, inflammatory boweldisease, Ulcerative colitis, Peyronie's Disease, coeliac disease,gallbladder disease, Pilonidal disease, peritonitis, psoriasis,vasculitis, surgical adhesions, stroke, Type I Diabetes, lyme arthritis,meningoencephalitis, immune mediated inflammatory disorders of thecentral and peripheral nervous system such as multiple sclerosis andGuillain-Barr syndrome, other autoimmune disorders, pancreatitis, trauma(surgery), graft-versus-host disease, transplant rejection, cancer (bothsolid tumours such as melanomas, hepatoblastomas, sarcomas, squamouscell carcinomas, transitional cell cancers, ovarian cancers andhematologic malignancies and in particular acute myelogenous leukaemia,chronic myelogenous leukemia, gastric cancer and colon cancer), heartdisease including ischaemic diseases such as myocardial infarction aswell as atherosclerosis, intravascular coagulation, bone resorption,osteoporosis, periodontitis and hypochlorhydia.

The present invention also provides an antibody molecule according tothe present invention for use in the treatment or prophylaxis of pain.

The present invention further provides the use of an antibody moleculeaccording to the present invention in the manufacture of a medicamentfor the treatment or prophylaxis of a pathological disorder that ismediated by IL-17A and/or IL-17F or associated with an increased levelof IL-17A and/or IL-17F. Preferably the pathological disorder isrheumatoid arthritis or multiple sclerosis.

The present invention further provides the use of an antibody moleculeaccording to the present invention in the manufacture of a medicamentfor the treatment or prophylaxis of pain.

An antibody molecule of the present invention may be utilised in anytherapy where it is desired to reduce the effects of IL-17A and/orIL-17F in the human or animal body. IL-17 A and/or IL-17F may becirculating in the body or may be present in an undesirably high levellocalised at a particular site in the body, for example a site ofinflammation.

An antibody molecule according to the present invention is preferablyused for the control of inflammatory disease, autoimmune disease orcancer.

The present invention also provides a method of treating human or animalsubjects suffering from or at risk of a disorder mediated by IL-17Aand/or IL-17F, the method comprising administering to the subject aneffective amount of an antibody molecule of the present invention.

An antibody molecule according to the present invention may also be usedin diagnosis, for example in the in vivo diagnosis and imaging ofdisease states involving IL-17A and/or IL-17F.

The present invention is further described by way of illustration onlyin the following examples, which refer to the accompanying Figures, inwhich:

FIG. 1 a) Light chain V region of antibody CA028_(—)0496 (SEQ ID NO:7)

-   -   b) Heavy chain V region of antibody CA028_(—)0496 (SEQ ID NO:9)    -   c) CDRH1 (SEQ ID NO:1), CDRH2 (SEQ ID NO:2), CDRH3 (SEQ ID        NO:3), CDRL1 (SEQ ID NO:4), CDRL2 (SEQ ID NO:5), CDRL3 (SEQ ID        NO:6) of antibody CA028_(—)496.    -   d) Light chain of antibody CA028_(—)496 (SEQ ID NO:11).    -   e) Heavy chain of antibody CA028496 (SEQ ID NO:15).    -   f) DNA encoding light chain of antibody CA028_(—)496 including        signal sequence (SEQ ID NO:14).    -   g) DNA encoding heavy chain of antibody CA028_(—)496 including        signal sequence (SEQ ID NO:18)

FIG. 2 a) The effect of antibody CA028_(—)0496 (designated Ab#496 inlegend) on human IL-17 induced IL-6 production from Hela cells. b) Theeffect of antibody CA028_(—)0496 (designated Ab#496 in legend) on humanIL-17F induced IL-6 production from Hela cells

DNA MANIPULATIONS AND GENERAL METHODS

E. coli strain INVαF′ (Invitrogen) was used for transformation androutine culture growth. DNA restriction and modification enzymes wereobtained from Roche Diagnostics Ltd. and New England Biolabs. Plasmidpreparations were performed using Maxi Plasmid purification kits(QIAGEN, catalogue No. 12165). DNA sequencing reactions were performedusing the ABI Prism Big Dye terminator sequencing kit (catalogue No.4304149) and run on an ABI 3100 automated sequencer (AppliedBiosystems). Data was analysed using the program AutoAssembler (AppliedBiosystems). Oligonucleotides were obtained from Invitrogen. Theconcentration of IgG was determined using IgG assembly ELISA.

IL-17 Isoforms

Recombinant IL-17A and IL-17F were purchased from R&D Systems.

Recombinant IL-17A/F heterodimer was produced by linking IL-17A andIL-17F using a GS linker. The heterodimer had the following sequence(SEQ ID NO:19)

MGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVAGGGGSGGGGSGGGGSGGGGSRKIPKVGHTFFQKPESCPPVPGGSMKLDIGIINENQRVSMSRNIESRSTSPWNYTVTWDPNRYPSEVVQAQCRNLGCINAQGKEDISMNSVPIQQETLVVRRKHQGCSVSFQLEKVLVTVGCTCVTPVIHHVQ

Recombinant cynomolgus IL-17F (SEQ ID NO:20)

MRKIPKVGHTFFQKPESCPPVPEGSMKLDTGIINENQRVSMSRNIESRSTSPWNYTVTWDPNRYPSEVVQAQCKHLGCINAQGKEDISMNSVPIQQETLVLRRKHQGCSVSFQLEKVLVTVGCTCVTPVIHHVQ

The DNA sequence encoding IL-17A/F heterodimer was chemicallysynthesised by Entelechon GmbH and was subcloned into pET43.1a at theNdeI/XhoI sites. The DNA sequence encoding cyno L-17F was amplified byPCR using primers that introduced NdeI and XhoI restriction sites. ThePCR products were ligated into pCR4Blunt-TOPO and sequence verifiedbefore digestion and ligation into pET43.1a at the NdeI/XhoI sites.

pET43.1a DNA encoding IL-17 isoforms was used to transfect BL21(DE3)cells and selected carbenicillin-resistant clones were grown at 37° C.overnight in 2TY broth containing 2% glucose and 50 μg/ml carbenicillin.The cultures were then diluted and grown in the same medium to an OD₆₀₀of 0.5-0.7, induced with 1 mM IPTG and grown at 37° C. for a further 4-5hours.

Cells were harvested by centrifugation and inclusion bodies preparedfrom the cells. Inclusion bodies were solubilised in 50 mM Tris-HCl, 5Mguanidinium hydrochloride, 50 mM NaCl, 1 mM EDTA, 2 mM reducedglutathione, 0.2 mM oxidised glutathione, pH 8.5. IL-17 protein wasrefolded by dropwise addition of the solubilised protein to the abovebuffer without guanidinium hydrochloride, with vigorous stirring. Thefinal volume was chosen such that the final protein concentration was nomore than 0.1 mg/ml.

The refolded protein solution was concentrated if required, beforebuffer exchange with 10 mM MES pH6. The protein was then applied to acolumn of Sepharose SP HP equilibrated with 20 mM MES pH6. Protein waseluted with a linear gradient of 0-500 mM NaCl in MES pH6 over 10 columnvolumes. For IL-17F the gradient was extended to 600 mM NaCl. In orderto further purify IL-17, the relevant fraction from the Sepharose SP HPcolumn were pooled, concentrated and diluted with 20 mM CAPSO (pH10) andapplied to a Mono Q column equilibrated with 20 mM CAPSO. Protein waseluted with a linear gradient of 0-250 mM NaCl in 20 mM CAPSO over 20column volumes. Fractions containing IL-17 were pooled and neutralisedusing 1M MES pH6.

EXAMPLE 1 Production of a Neutralising Anti-IL-17 Antibody

Female Sprague Dawly rats were immunised with recombinant human IL-17(purchased from R & D systems). Rats received four immunisations of 20μg IL-17 in 1000 Freund's adjuvant. Antibody 225 which binds human IL-17was isolated using the methods described in WO04/051268. Genes for theheavy chain variable domain (VH) and light chain variable domain (VL) ofantibody 225 were isolated and sequenced following cloning via reversetranscription PCR.

A series of humanised VL and VH regions were designed using humanV-region acceptor frameworks and by varying the number of donor residuesin the framework regions. Eight grafted VL regions (gL1-8) and 9 graftedVH regions (gH1-9) were designed and genes were built by oligonucleotideassembly and PCR mutagenesis.

The light chain grafted sequences were sub-cloned into the human lightchain expression vector pKH10.1 which contains the DNA encoding thehuman C-Kappa constant region (Km3 allotype). The heavy chain graftedsequences were sub-cloned into the human gamma-4 expression vectorpVhg4P FL, which contains the DNA encoding the human gamma-4 constantregion containing the hinge stabilising mutation S241P (Angal et al.,supra). Plasmids were co-transfected into CHO cells and the antibodiesproduced screened for activity in IL-17 binding and neutralisationassays. Transfections of CHO cells were performed using theLipofectamine™ 2000 procedure according to manufacturer's instructions(InVitrogen, catalogue No. 11668).

The most optimal graft based on expression, affinty and neutralisationpotency (gL7gH9) was selected and named CA028_(—)0496. The V regionsequences of this antibody are shown in FIGS. 1 (a) and (b) and in SEQID NOs: 7 and 9 for the light chain (gL7) and heavy chains (gH9)respectively.

The heavy chain acceptor framework is the human germline sequence VH31-3 3-07 with framework 4 coming from this portion of the humanJH-region germline JH4. The light chain acceptor framework is the humangermline sequence VK1 2-1-(1) L4, with framework 4 coming from thisportion of the human JK-region germline JK1.

EXAMPLE 2 Antibody CA028_(—)0496 Neutralises IL-17 and IL-17F andIL-17A/F Heterodimer

Hela Cells

The potency of antibody CA028_(—)0496 against human recombinant IL-17and human recombinant IL-17F in Hela cells was tested and compared toantibody CDP435 (WO06/054059). Hela cells were obtained from the cellbank at ATCC (ATCC CCL-2). Cells were grown in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% foetal calf serum,penicillin, gentamycin and glutamine. 1×10⁴ cells were plated out into96 well flat bottomed tissue culture plates. Cells were incubatedovernight and washed once in assay buffer. Either human IL-17A (25 ngml⁻¹) or human IL-17F (125 ng ml⁻¹) was incubated in the presence of afixed concentration of human TNF-α this mixture was preincubated withantibody CA028_(—)0496 or antibody CDP435. Cytokine plus antibody wasthen added to the Hela cells which were incubated overnight. Theproduction of IL-6 in the cell culture supernatant was proportionate tothe amount of IL-17A/IL-17F added to the cells. Human IL-6 levels weremeasured by ELISA and quantified by comparison with known standardconcentrations of human IL-6.

The data (FIGS. 2 a and 2 b) indicates that antibody CA028_(—)0496potently neutralised human recombinant IL-17A and also had some activityagainst human IL-17F. The data from these experiments indicated thatantibody CA028_(—)0496 gave an IC₅₀ of 43/ng/ml against humanrecombinant IL-17 (25 ng ml⁻¹) and 1477 ng/ml against recombinant IL-17F(125 ng ml⁻¹).

Accordingly, antibody CA028_(—)0496 gave an IC50 of 0.29M against humanrecombinant IL-17 (0.78 nM) and 10.18 nM against human recombinantIL-17F (4.16 nM) in this assay (calculation based on per IgG assuming amolecular weight of 145,000 as an average IgG4 and assuming that IL-17Aand IL-17F are dimers).

Human Microglia Cells

Human microglia cells (TCS Cellworks) were plated out in a flat bottom96-well plate at 5,000 cells per well in a total volume of 100 μl andleft for 24 hours to attach to the plastic. At this time titrations (5,1, 0.2 and 0.04 μg/ml) of human recombinant IL-17A, human recombinantIL-17F, cynomolgus recombinant IL-17F and human recombinant IL-17A/Fheterodimer in the presence and absence of 10 ng/ml human recombinantTNFα were added to wells in triplicate. Control wells contained nostimulation, IL-17A alone (100 ng/ml), TNFα alone and IL-17A and TNFαtogether. All cytokines were added in a total volume of 110 μl/well,making the total well volume 210 μl. In experiments involvingantibodies, cells were plated out in the same way. After 24 hoursantibodies and cytokines were added at the same time to give the statedfinal concentrations in a total final volume of 200 μl.

After a further 24 hours incubation at 37° C., supernatants wereharvested and frozen at −20° C. until analysis. For analysis,supernatants were diluted 1/10 and measured for IL-6 using a human IL-6MSD kit, according to manufacturer's instructions.

All isoforms of IL-17 tested were found to be active in the assay,particularly in the presence of TNFα.

The potency of antibody CA028_(—)0496 against human recombinant IL-17Aand human recombinant IL-17F, cynomolgus recombinant IL-17F and humanrecombinant IL-17A/F heterodimer in human microglia cells was tested inthe presence of TNFα and compared to a control antibody and an IL-17Aspecific antibody using the method described above.

The control antibody had no effect on the activity of any of thecytokines tested. Antibody CA028_(—)0496 had inhibitory activity againstall three cytokines IL-17, IL-17F and IL-17A/F, including cynomolgusIL-17F while the IL-17A specific antibody only had inhibitory activityagainst IL-17A and IL-17A/F heterodimer.

EXAMPLE 3 Affinity of Antibody CA028_(—)0496 (Human IgG4 ConstantRegions) for IL-17A and IL-17F

BIA (Biamolecular Interaction Analysis) was performed using a Biacore3000 (Biacore AB). All experiments were performed at 25° C. AffinipureFc Fragment goat anti-human IgG, Fc fragment specific (JacksonImmunoResearch) was immobilised on a CM5 Sensor Chip via amine couplingchemistry to a capture level of ≈6000 response units (RUs). HBS-EPbuffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% SurfactantP20, Biacore AB) was used as the running buffer with a flow rate of 10μl/min. A 10 μl injection of antibody CA028_(—)0496 (1.81 mg/ml) wasused for capture by the immobilised anti-human IgG-Fc. Human IL-17A andIL-17 isoforms were titrated over the captured CA028_(—)0496 at doublingdilutions from 50 nM to sub nM at a flow rate of 30 μL/min. The surfacewas regenerated by a 30 μL injections of 40 mM HCl, followed by one 5 μLinjection of 5 mM NaOH.

Background subtraction binding curves were double referenced andanalysed using the BIAevaluation software (version 3.2) followingstandard procedures. Kinetic parameters were determined from the fittingalgorithm.

The affinity value determined for antibody CA028_(—)0496 binding IL-17Awas 16 pM and 1750 pM for IL-17F. Antibody CA028_(—)0496 did not bind tothe other IL-17 isoforms (IL-17 B, C, D and E). Antibody CA028_(—)0496therefore specifically binds IL-17A and IL-17F.

EXAMPLE 4 Affinity of Antibody CA028_(—)0496 (Murine IgG1 ConstantRegions) for IL-17A, Cynomolgus IL-17F and IL-17A/F Heterodimer

BIA (Biamolecular Interaction Analysis) was performed using a Biacore3000 (Biacore AB).

All experiments were performed at 25° C. Affinipure F(ab′)₂ fragmentgoat anti-mouse IgG, Fc fragment specific (Jackson ImmunoResearch) wasimmobilised on a CM5 Sensor Chip (Biacore AB) via amine couplingchemistry to a capture level of ≈6000 response units (RUs). HBS-EPbuffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% SurfactantP20, Biacore AB) was used as the running buffer with a flow rate of 10μL/min. A 10 μL injection of antibody CA028_(—)0496 at 4 ug/mL was usedfor capture by the immobilised anti-mouse IgG, Fc. Human IL-17A, cynoIL-17F and heterodimerA/F were titrated over the captured CA028_(—)0496at doubling dilutions from 25 nM to sub nM at a flow rate of 30 μL/min.The surface was regenerated at a flowrate of 10 uL/min by a 10 μLinjection of 40 mM HCl, followed by a 5 μL injection of 5 mM NaOH.

Double referenced background subtracted binding curves were analysedusing the BIAevaluation software (version 3.2) following standardprocedures. Kinetic parameters were determined from the fittingalgorithm.

Antibody CA028_(—)0496 had an affinity of 21 pM for IL-17A, 116 pM forIL-17A/F heterodimer and 1030 pM for cynomolgus IL-17F.

It will of course be understood that the present invention has beendescribed by way of example only, is in no way meant to be limiting, andthat modifications of detail can be made within the scope of the claimshereinafter. Preferred features of each embodiment of the invention areas for each of the other embodiments mutatis mutandis. All publications,including but not limited to patents and patent applications, cited inthis specification are herein incorporated by reference as if eachindividual publication were specifically and individually indicated tobe incorporated by reference herein as though fully set forth.

The invention claimed is:
 1. An isolated DNA molecule encoding aneutralising antibody which binds human IL-17A and human IL-17F, whereinthe variable domain of the heavy chain comprises the sequence given inSEQ ID NO: 1 for CDR-H1, the sequence given in SEQ ID NO: 2 for CDR-H2and the sequence given in SEQ ID NO: 3 for CDR-H3, and wherein thevariable domain of the light chain comprises the sequence given in SEQID NO: 4 for CDR-L1, the sequence given in SEQ ID NO: 5 for CDR-L2 andthe sequence given in SEQ ID NO: 6 for CDR-L3, or an antigen-bindingfragment thereof.
 2. A cloning or expression vector comprising the DNAmolecule according to claim
 1. 3. The vector according to claim 2,wherein the vector comprises the sequences given in SEQ ID NO: 14 andSEQ ID NO:
 18. 4. An isolated host cell comprising the vector accordingto claim
 3. 5. A process for the production of an antibody encoded bythe DNA molecule in the vector of the isolated host cell of claim 4,comprising culturing the isolated host cell under conditions suitablefor expression of the antibody, and isolating the antibody.
 6. Anisolated host cell comprising the vector according to claim
 2. 7. Aprocess for the production of an antibody encoded by the DNA molecule inthe vector of the isolated host cell of claim 6, comprising culturingthe isolated host cell under conditions suitable for expression of theantibody, and isolating the antibody.
 8. An isolated DNA moleculeencoding a neutralising antibody which binds human IL-17A and humanIL-17F, wherein the heavy chain comprises the sequence given in SEQ IDNO: 9 and wherein the light chain comprises the sequence given in SEQ IDNO: 7, or an antigen-binding fragment thereof.
 9. A cloning orexpression vector comprising the DNA molecule according to claim
 8. 10.An isolated host cell comprising the vector according to claim
 9. 11. Aprocess for the production of an antibody encoded by the DNA molecule inthe vector of the isolated host cell of claim 10, comprising culturingthe isolated host cell under conditions suitable for expression of theantibody, and isolating the antibody.
 12. An isolated DNA moleculeencoding a neutralising antibody which binds human IL-17A and humanIL-17F, wherein the heavy chain comprises the sequence given in SEQ IDNO: 15 and wherein the light chain comprises the sequence given in SEQID NO: 11, or an antigen-binding fragment thereof.
 13. A cloning orexpression vector comprising the DNA molecule according to claim
 12. 14.An isolated host cell comprising the vector according to claim
 13. 15. Aprocess for the production of an antibody encoded by the DNA molecule inthe vector of the isolated host cell of claim 14, comprising culturingthe isolated host cell under conditions suitable for expression of theantibody, and isolating the antibody.
 16. An isolated DNA moleculeencoding a neutralising antibody which binds human IL-17A and humanIL-17F, wherein the heavy chain comprises the sequence given in SEQ IDNO:
 9. 17. An isolated DNA molecule encoding a neutralising antibodywhich binds human IL-17A and human IL-17F, wherein the light chaincomprises the sequence given in SEQ ID NO: 7.